WO2006128902A1 - Proteines slc39a12 en tant que cibles diagnostiques et therapeutiques pour les maladies neurodegeneratives - Google Patents

Proteines slc39a12 en tant que cibles diagnostiques et therapeutiques pour les maladies neurodegeneratives Download PDF

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WO2006128902A1
WO2006128902A1 PCT/EP2006/062835 EP2006062835W WO2006128902A1 WO 2006128902 A1 WO2006128902 A1 WO 2006128902A1 EP 2006062835 W EP2006062835 W EP 2006062835W WO 2006128902 A1 WO2006128902 A1 WO 2006128902A1
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slc39a12
seq
proteins
disease
expression
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Johannes Pohlner
Heinz Von Der Kammer
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Evotec Neurosciences GmbH
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Evotec Neurosciences GmbH
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Priority to JP2008514110A priority Critical patent/JP2008545424A/ja
Priority to US11/921,070 priority patent/US20090133135A1/en
Priority to EP06763457.6A priority patent/EP1891234B1/fr
Publication of WO2006128902A1 publication Critical patent/WO2006128902A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present invention relates to methods of diagnosing, prognosticating and monitoring the progression of neurodegenerative diseases in a subject. Furthermore, methods of therapy control and screening for modulating agents of neurodegenerative diseases are provided. The invention also discloses pharmaceutical compositions, kits, and recombinant animal models.
  • AD Alzheimer's disease
  • AD Alzheimer's disease
  • these diseases constitute an enormous health, social, and economic burden.
  • AD is the most common neurodegenerative disease, accounting for about 70 % of all dementia cases, and it is probably the most devastating age-related neurodegenerative condition affecting about 10 % of the population over 65 years of age and up to 45 % over age 85 (Vickers et al., Progress in Neurobiology 2000, 60: 139-165; Walsh and Selkoe, Neuron 2004, 44: 181-193).
  • the neuropathological hallmarks that occur in the brains of individuals with AD are senile plaques, composed of amyloid- ⁇ protein, and profound cytoskeletal changes coinciding with the appearance of abnormal filamentous structures and the formation of neurofibrillary tangles.
  • the amyloid- ⁇ protein evolves from the cleavage of the amyloid precursor protein (APP) by different kinds of proteases (Selkoe and Kopan, Annu Rev Neurosci 2003, 26:565-597; Ling et al., lnt J Biochem Cell Biol 2003, 35:1505- 1535).
  • Two types of plaques, diffuse plaques and neuritic plaques can be detected in the brain of AD patients. They are primarily found in the cerebral cortex and hippocampus.
  • the present invention is based on the finding of the association of the metal ion transporter solute carrier family 39 member 12, SLC39A12, with neurodegenerative diseases, in particular Alzheimer's disease.
  • neurodegenerative diseases in particular Alzheimer's disease.
  • Besides a number of biological functions throughout the whole human body zinc plays an important role in the function of neurons either by modulating protein function or as ionic signal (Frederickson et al., Nature Rev Neurosci. 2005, AOP, doi:10.1038/nrn1671 ).
  • Specifically synaptic release Of Zn 2+ neurons is described to be synchronistic with the release of glutamate.
  • this specific subpopulation of neurons mainly located with their cell bodies within the cerebral cortex and the limbic structures, is also termed as "gluzinerg” (Slomianka et al., Neuroscience 1990, 38:843-854; Frederickson & Bush, BioMetals 2001 , 14:353-366).
  • Active and/or passive transport of zinc required for the regulation of zinc homeostasis is accomplished by zinc transporters which are members of the solute carrier (SLC) gene series (Liuzzi and Cousins, Annu. Rev. Nutr. 2004, 24:151 -172).
  • SLC solute carrier
  • Figure 1 discloses the identification of differences in the levels of SLC39A12 gene derived mRNA in human brain tissue samples from individuals corresponding to different Braak stages as measured and compared by GeneChip analyses. It indicates that the levels of the respective mRNA species correlate quantitatively with AD progression and thus are indicative for AD as measured by the neuropathological staging of brain tissue samples according to Braak and Braak (Braak staging).
  • Figure 2 lists the data for the verification of differences in the levels of SLC39A12 gene derived mRNA in human brain tissue samples from individuals corresponding to different Braak stages indicative for AD as measured by quantitative RT-PCR analysis.
  • Figure 4C discloses SEQ ID NO: 3, the amino acid sequence of the human SLC39A12 splice variant 3 protein.
  • Figure 5C shows SEQ ID NO: 6, the nucleotide sequence of the human SLC39A12 splice variant 3 cDNA.
  • Figure 6A depicts SEQ ID NO: 7, the coding sequence (cds) of the human SLC39A12 splice variant 1.
  • Figure 6B depicts SEQ ID NO: 8, the coding sequence (cds) of the human SLC39A12 splice variant 2.
  • Figure 6C depicts SEQ ID NO: 9, the coding sequence (cds) of the human SLC39A12 splice variant 3.
  • Figure 7 depicts the sequence alignment of the primers used for SLC39A12 transcription level profiling by quantitative RT-PCR with the corresponding clippings of SLC39A12 cDNA.
  • Figure 9 shows an immunoblot (Western blot) analysis of the affinity-purified polyclonal rabbit anti-SLC39A12 antiserum HKQ1.
  • Figure 10 shows an immunofluorescense analysis of the affinity-purified polyclonal rabbit anti-SLC39A12 antiserum HKQ1.
  • Figures 11 A exemplifies the increase in the level of SLC39A12 protein in brain cerebral cortex tissue samples from AD patients (Braak 4-6) when compared to the levels observed in respective samples from age-matched controls (Braak 1 - 3) which have not been diagnosed to suffer from AD signs and symptoms.
  • Figures 11 B exemplifies the increase in the level of SLC39A12 protein in brain cerebral white matter tissue samples from AD patients (Braak 4 and 5) when compared to the levels observed in respective samples from age-matched controls (Braak 0 and 2) which have not been diagnosed to suffer from AD signs and symptoms.
  • Figure 12 shows detection of SLC39A12 protein expressed by three independent SLC39A12 transgenic fly lines under the control of gmr-GAL4 by Western blots analysis.
  • Figure 13 shows a comparison of SLC39A12 mRNA level expressed by three independent SLC39A12 transgenic fly lines under the control of gmr-GAL4 by RT-PCR using SLC39A12 specific primers.
  • FIG 14 shows that co-expression of SLC39A12 in TauP301 L transgenic Drosophila results in a significant and dosage dependent increase in the phosphorylation of Ser214 of mutant tau protein in comparison to control flies (TauP301 L).
  • Figure 15 shows SLC39A12-expression in H4-neuroglioma cells stably expressing the Swedish Mutant APP by Western blot analysis.
  • FIG 16 shows SLC39A12 expression in H4-neuroglioma cells stably expressing the Swedish Mutant APP by immunofluorescence analysis.
  • Figure 17 shows the verification of the function of SLC39A12 as zinc transporter by using a zinc uptake assay.
  • SLC39A12 when overexpressed in H4-cells, causes an increase in the intracellular concentration of zinc.
  • This cell based assay provides a means for the identification of small molecule based modulators of SLC39A12, .
  • activity shall be understood as a measure for the ability of a substance, such as transcription product or a translation product to produce a biological effect or a measure for a level of biologically active molecules.
  • activity also refers to biological activity and/or pharmacological activity which refer to binding, antagonization, repression, blocking, neutralization or sequestration of a transporter or transporter subunit and which refers to activation, agonization, and up-regulation of a transporter or transporter subunit.
  • modulator refers to a molecule capable of changing or altering the level and/or the activity of a gene, or a transcription product of a gene, or a translation product of a gene.
  • a “modulator” refers to a molecule which has the capacity to either enhance or inhibit, thus to “modulate” a functional property of a protein, to “modulate” binding, antagonization, repression, blocking, neutralization or sequestration, activation, agonization and up-regulation.
  • Modulation will be also used to refer to the capacity to affect the biological activity of a cell.
  • a “modulator” is capable of changing or altering the biological activity of a transcription product or a translation product of a gene. Said modulation, for instance, may be an increase or a decrease in the biological activity and/or pharmacological activity, a change in binding characteristics, or any other change or alteration in the biological, functional, or immunological properties of said translation product of a gene.
  • oligonucleotide primer or “primer” refer to short nucleic acid sequences which can anneal to a given target polynucleotide by hybridization of the complementary base pairs and can be extended by a polymerase. They may be chosen to be specific to a particular sequence or they may be randomly selected, e.g. they will prime all possible sequences in a mix. The length of primers used herein may vary from 10 nucleotides to 80 nucleotides.
  • Probes are short nucleic acid sequences of the nucleic acid sequences described and disclosed herein or sequences complementary therewith. They may comprise full length sequences, or fragments, derivatives, isoforms, or variants of a given sequence. The identification of hybridization complexes between a "probe” and an assayed sample allows the detection of the presence of other similar sequences within that sample.
  • variant refers to any polypeptide or protein, in reference to polypeptides and proteins disclosed in the present invention, in which one or more amino acids are added and/or substituted and/or deleted and/or inserted at the N-terminus, and/or the C-terminus, and/or within the native amino acid sequences of the native polypeptides or proteins of the present invention, but retains its essential properties.
  • variant refers to any mRNA, in reference to gene transcripts disclosed in the present invention, in which one or more nucleotides are added and/or substituted and/or deleted.
  • Proteins and polypeptides of the present invention include variants, fragments and chemical derivatives of the proteins comprising the amino acid sequences of SLC39A12 proteins having SEQ ID NO: 1 , or SEQ ID NO: 2, or SEQ ID NO: 3. Included shall be such exchange of amino acids which would have no effect on functionality, such as arginine for lysine, valine for leucine, asparagine for glutamine. Proteins and polypeptides can be included which can be isolated from nature or be produced by recombinant and/or synthetic means. Native proteins or polypeptides refer to naturally-occurring truncated or secreted forms, naturally occurring variant forms (e.g. splice-variants) and naturally occurring allelic variants.
  • isolated as used herein is considered to refer to molecules or substances which have been changed and/or that are removed from their natural environment, i.e. isolated from a cell or from a living organism in which they normally occur, and that are separated or essentially purified from the coexisting components with which they are found to be associated in nature.
  • sequences encoding such molecules can be linked by the hand of man to polynucleotides, to which they are not linked in their natural state and such molecules can be produced by recombinant and/or synthetic means, it is also said that they are "non-native".
  • AD shall mean Alzheimer's disease.
  • AD-type neuropathology refers to neuropathological, neurophysiological, histopathological and clinical hallmarks, signs and symptoms as described in the instant invention and as commonly known from state-of-the-art literature (see: Iqbal, Swaab, Winblad and Wisniewski, Alzheimer ' s Disease and Related Disorders (Etiology, Pathogenesis and Therapeutics), Wiley & Sons, New York, Weinheim, Toronto, 1999; Scinto and Daffner, Early Diagnosis of Alzheimer ' s Disease, Humana Press, Totowa, New Jersey, 2000; Mayeux and Christen, Epidemiology of Alzheimer ' s Disease: From Gene to Prevention, Springer Press, Berlin, Heidelberg, New York, 1999; Younkin, Tanzi and Christen, Presenilins and Alzheimer ' s Disease, Springer Press, Berlin, Heidelberg, New York, 1998).
  • Brain stage or “Braak staging” refers to the classification of brains according to the criteria proposed by Braak and Braak (Braak and Braak, Acta Neuropathology 1991 , 82: 239-259).
  • Braak staging of AD rates the extent and distribution of neurofibrillary pathology in determined regions of the forebrain and divides the neuropathologic progression of AD into six stages (stage 0 to 6). It is a well established and universally accepted procedure in post-mortem neuropathological staging of AD.
  • Braak stages are therefore used as a surrogate marker of disease progression independent of the clinical presentation/condition of the individual donor, i.e. independent of the presence or absence of reported mental illness, cognitive deficits, decline in other neuropsychiatric parameters, or the overt clinical diagnosis of AD.
  • the neurofibrillary changes on which the Braak staging reflect the underlying molecular and cellular pathomechanisms in general and hence define a (pre-)morbid condition of the brain, meaning that e.g. a donor staged Braak 1 represents by definition an earlier stage of molecular/cellular pathogenesis than a donor staged 2 (or higher), and that therefore a donor of Braak stage 1 can e.g.
  • control individual and affected individual may not necessarily be the same as the clinical diagnosis based differentiation between healthy control donor and AD patient, but it rather refers to a presumed difference in the (pre-) morbid status as deduced from and mirrored by a surrogate marker, the Braak stage.
  • SLC39A12 may be causally involved in the cascade of molecular pathological events leading to AD and therefore may represent a promising target for the identification and development of small molecule based therapeutics for AD and other neurodegenerative diseases.
  • the present invention discloses a dysregulation of a gene coding for SLC39A12 and of its gene products in specific brain regions of AD patients.
  • Neurons within the inferior temporal lobe, the entorhinal cortex, the hippocampus, and the amygdala are subject to degenerative processes in AD (Terry et al., Annals of Neurology 1981 , 10: 184-192).
  • These brain regions are mostly involved in the processing of learning and memory functions and display a selective vulnerability to neuronal loss and degeneration in AD.
  • neurons within the frontal cortex, the occipital cortex, and the cerebellum remain largely intact and preserved from neurodegenerative processes.
  • Brain tissues from the frontal cortex (F) and the inferior temporal cortex (T) of AD patients and of age- matched controls were used for the herein disclosed examples. Consequently, the SLC39A12 gene and its corresponding transcription and/or translation products play a causative role, and/or have an influence on the selective neuronal degeneration and/or neuroprotection.
  • the invention features a method of diagnosing or prognosticating a neurodegenerative disease in a subject, or of determining whether a subject has a predisposition of developing said disease, is at increased risk of developing said disease, or of monitoring the effect of a treatment administered to a subject having a neurodegenerative disease.
  • the method comprises: determining a level, an expression or an activity, or both said level, expression and said activity of (i) a transcription product of a gene coding for SLC39A12 proteins, and/or of (ii) a translation product of a gene coding for SLC39A12 proteins, and/or of (iii) a fragment, or derivative, or variant of said transcription or translation product in a sample obtained from said subject and comparing said level, expression and/or said activity of said transcription product and/or said translation product and/or said fragment, derivative or variant thereof to a reference value representing a known disease status (patient) and/or to a reference value representing a known health status (control), and/or to a reference value representing a known Braak stage and analysing whether said level and/or said activity is varied, is altered compared to a reference value representing a known health status, and/or is similar or equal to a reference value representing a known disease status and/or is similar compared to a reference value representing a known Braak stage which is an indication
  • the invention features a method of monitoring the progression of a neurodegenerative disease in a subject.
  • a level, expression or an activity, or both said level, expression and said activity, of (i) a transcription product of a gene coding for SLC39A12 proteins, and/or of (ii) a translation product of a gene coding for SLC39A12 proteins, and/or of (iii) a fragment, or derivative, or variant of said transcription or translation product in a sample obtained from said subject is determined.
  • Said level, expression and/or said activity are compared to a reference value representing a known disease or health status or a known Braak stage. Thereby, the progression of said neurodegenerative disease in said subject is monitored.
  • the invention features a method of evaluating a treatment or monitoring the effect of a treatment for a neurodegenerative disease, comprising determining a level, expression or an activity, or both said level, expression and said activity of (i) a transcription product of a gene coding for SLC39A12 proteins, and/or of (ii) a translation product of a gene coding for SLC39A12 proteins, and/or of (iii) a fragment, or derivative, or variant of said transcription or translation product in a sample obtained from a subject being treated for said disease. Said level, expression or said activity, or both said level, expression and said activity are compared to a reference value representing a known disease or health status or a known Braak stage, thereby evaluating the treatment for said neurodegenerative disease.
  • the level, expression or the activity, or both said level and said activity of (i) a transcription product of a gene coding for SLC39A12 proteins, and/or of (ii) a translation product of a gene coding for SLC39A12 proteins, and/or of (iii) a fragment, or derivative, or variant of said transcription or translation product in a series of samples taken from said subject over a period of time is compared, in order to monitor the progression of said disease.
  • said subject receives a treatment prior to one or more of said sample gatherings.
  • said level and/or activity is determined before and after said treatment of said subject.
  • said level, the expression and/or said activity of said transcription product and/or said translation product of SLC39A12 and of its fragments, derivatives, or variants is increased, is up-regulated in samples obtained from AD patients as compared to samples obtained from persons not suffering from AD, control persons.
  • the expression and/or activity of the transcription product and/or the translation product of SLC39A12 and of its fragments, derivatives, or variants is measured from samples of patients and compared with the expression and/or activity of the transcription product and/or the translation product of SLC39A12 and of its fragments, derivatives, or variants in a sample of a healthy control subject (reference sample).
  • said SLC39A12 gene codes for proteins having SEQ ID NO: 1 (splice variant 1 (sv1 ), Genbank/Ensembl accession number BC094700/ENST00000377369), or SEQ ID NO: 2 (splice variant 2 (sv2), Genbank/Ensembl accession number Q5VWV9,BC035118/ENST00000377371 ), or SEQ ID NO: 3 (splice variant 3 (sv3), Genbank/Ensembl accession number Q5VWV8/ ENST00000277633).
  • sequences are "isolated" as the term is employed herein.
  • the gene coding for said SLC39A12 proteins (splice variants sv1 , sv2, and sv3) is also generally referred to as the SLC39A12 gene or simply SLC39A12.
  • the proteins (splice variants sv1 , sv2, and sv3) of SLC39A12 are also generally referred to as the SLC39A12 proteins, SLC39A12 splice variants or simply SLC39A12.
  • said neurodegenerative disease or disorder is Alzheimer's disease, and said subjects suffer from signs and symptoms of Alzheimer's disease.
  • said reference value is that of a level, of expression, or of an activity, or both of said level and said activity of (i) a transcription product of the gene coding for SLC39A12 proteins, and/or of (ii) a translation product of the gene coding for SLC39A12 proteins, and/or of (iii) a fragment, or derivative, or variant of said transcription or translation product in a sample obtained from a subject not suffering from said neurodegenerative disease (control sample, control, healthy control person) or in a sample obtained from a subject suffering from a neurodegenerative disease, in particular
  • Alzheimer's disease patient sample, patient, AD sample
  • a person with a defined Braak stage which may suffer or may not suffer from signs and symptoms of AD.
  • an alteration in the level and/or activity and/or expression of a transcription product of the gene coding for SLC39A12 proteins and/or of a translation product of the gene coding for SLC39A12 proteins and/or of a fragment, or derivative, or variant thereof in a sample cell, or tissue, or body fluid taken from said subject relative to a reference value representing a known health status (control sample) indicates a diagnosis, or prognosis, or increased risk of becoming diseased with a neurodegenerative disease, particularly AD.
  • said varied, altered level, altered expression and/or said altered activity of said transcription product and/or said translation product of SLC39A12 and of its fragments, derivatives, or variants is an increase, an up-regulation.
  • the invention also relates to the construction and the use of primers and probes which are unique to the nucleic acid sequences, or fragments, or variants thereof, as disclosed in the present invention.
  • the oligonucleotide primers and/or probes can be labeled specifically with fluorescent, bioluminescent, magnetic, or radioactive substances.
  • the invention further relates to the detection and the production of said nucleic acid sequences, or fragments and variants thereof, using said specific oligonucleotide primers in appropriate combinations.
  • PCR-analysis a method well known to those skilled in the art, can be performed with said primer combinations to amplify said gene specific nucleic acid sequences from a sample containing nucleic acids.
  • Such sample may be derived either from healthy or diseased subjects or subjects with defined Braak stages. Whether an amplification results in a specific nucleic acid product or not, and whether a fragment of different length can be obtained or not, may be indicative for a neurodegenerative disease, in particular Alzheimer's disease.
  • the invention provides nucleic acid sequences, oligonucleotide primers, and probes of at least 10 bases in length up to the entire coding and gene sequences, useful for the detection of gene mutations and single nucleotide polymorphisms in a given sample comprising nucleic acid sequences to be examined, which may be associated with neurodegenerative diseases, in particular Alzheimer's disease.
  • This feature has utility for developing rapid DNA- based diagnostic tests, preferably also in the format of a kit.
  • Primers for SLC39A12 are exemplarily described in Example 1 (vi).
  • the invention features a kit for diagnosing or prognosticating neurodegenerative diseases, in particular AD, in a subject, or determining the propensity or predisposition of a subject to develop a neurodegenerative disease, in particular AD, or of monitoring the effect of a treatment administered to a subject having a neurodegenerative disease, particularly AD, said kit comprising:
  • the kit may be particularly useful for the identification of individuals that are at risk of developing a neurodegenerative disease, in particular AD.
  • Reagents that selectively detect a transcription product and/or a translation product of the gene coding for SLC39A12 proteins, preferably coding for the splice variants having SEQ ID NO: 1 ,or having SEQ ID NO: 2, or having SEQ ID NO: 3 can be sequences of various length, fragments of sequences, antibodies, aptamers, siRNA, microRNA, ribozymes. Such reagents may be used also to detect fragments, derivatives or variants thereof.
  • the kit may serve as a means for targeting identified individuals for early preventive measures or therapeutic intervention prior to disease onset, before irreversible damage in the course of the disease has been inflicted.
  • the kit featured in the invention is useful for monitoring a progression of a neurodegenerative disease, in particular AD in a subject, as well as monitoring success or failure of therapeutic treatment for such a disease of said subject.
  • the invention features a method of treating or preventing a neurodegenerative disease, in particular AD, in a subject comprising the administration to said subject in need of such a treatment in a therapeutically or prophylactically effective amount and formulation an agent, agents, modulators, antagonist, agonists or antibodies which directly or indirectly affect a level, or an activity, or both said level and said activity, of (i) the gene coding for SLC39A12 proteins, and/or (ii) a transcription product of the gene coding for SLC39A12 proteins, and/or (iii) a translation product of the gene coding for SLC39A12 proteins, and/or (iv) a fragment, or derivative, or variant of (i) to (iii).
  • an agent, agents, modulators, antagonist, agonists or antibodies which directly or indirectly affect a level, or an activity, or both said level and said activity, of (i) the gene coding for SLC39A12 proteins, and/or (ii) a transcription product of the gene coding for SLC
  • Said agent may comprise a small molecule, or it may also comprise a peptide, an oligopeptide, or a polypeptide.
  • Said peptide, oligopeptide, or polypeptide may comprise an amino acid sequence of a translation product of the gene coding for SLC39A12 proteins, or a fragment, or derivative, or a variant thereof.
  • An agent for treating or preventing a neurodegenerative disease, in particular AD, according to the instant invention may also consist of a nucleotide, an oligonucleotide, or a polynucleotide.
  • Said oligonucleotide or polynucleotide may comprise a nucleotide sequence of the gene coding for SLC39A12 proteins, either in sense orientation or in antisense orientation.
  • RNA interference RNA interference
  • the method comprises grafting donor cells into the central nervous system, preferably the brain, of said subject, or donor cells preferably treated so as to minimize or reduce graft rejection, wherein said donor cells are genetically modified by insertion of at least one transgene encoding said agent or agents.
  • Said transgene might be carried by a viral vector, in particular a retroviral vector.
  • the transgene can be inserted into the donor cells by a nonviral physical transfection of DNA encoding a transgene, in particular by microinjection.
  • the subject for treatment or prevention can be a human, or a non-human experimental animal, e.g. a mouse or a rat, a domestic animal, or a non-human primate.
  • the experimental animal can be an animal model for a neurodegenerative disorder, e.g. a transgenic mouse and/or a knock-out mouse with an AD-type neuropathology.
  • the invention features a pharmaceutical composition
  • a pharmaceutical composition comprising said agent, antibody, antagonist or agonist, or modulator and preferably a pharmaceutical carrier.
  • Said carrier refers to a diluent, adjuvant, excipient, or vehicle with which the modulator is administered.
  • the present invention also provides a kit comprising one or more containers filled with a therapeutically or prophylactically effective amount of said pharmaceutical composition.
  • the generation of said recombinant, non-human animal comprises (i) providing a gene targeting construct containing said gene sequence and a selectable marker sequence, and (ii) introducing said targeting construct into a stem cell of a non-human animal, and (iii) introducing said non- human animal stem cell into a non-human embryo, and (iv) transplanting said embryo into a pseudopregnant non-human animal, and (v) allowing said embryo to develop to term, and (vi) identifying a genetically altered non-human animal whose genome comprises a modification of said gene sequence in both alleles, and (vii) breeding the genetically altered non-human animal of step (vi) to obtain a genetically altered non-human animal whose genome comprises a modification of said gene sequence, wherein the expression of said gene, a mis-expression, under-expression, non-expression or over-expression, and wherein the disruption or alteration of said gene sequence results in said non-human animal exhibiting a predisposition to developing signs and symptoms of a neurode
  • Said method may be useful for the identification of novel compounds as well as for evaluating compounds which have been improved or otherwise optimized in their ability to inhibit the binding of a ligand to a gene product of the gene coding for SLC39A12 protein, or a fragment, or derivative, or variant thereof.
  • a fluorescent binding assay in this case based on the use of carrier particles, is disclosed and described in patent application WO00/52451.
  • a further example is the competitive assay method as described in patent WO02/01226.
  • the present invention provides a method for producing a medicament comprising the steps of (i) identifying a compound as an inhibitor of binding between a ligand and a gene product of the gene coding for SLC39A12 proteins by the aforementioned inhibitory binding assay and (ii) admixing the compound with a pharmaceutical carrier.
  • said compound may also be identifiable by other types of screening assays.
  • the present invention provides an assay for testing a compound or compounds, preferably for screening a plurality of compounds in high- throughput format to determine the degree of binding of said compounds to SLC39A12 protein (preferably having SEQ ID NO: 1 , or SEQ ID NO: 2, or SEQ ID NO: 3), or to a fragment, or derivative, or variant thereof
  • said screening assay comprises (i) adding a liquid suspension of said SLC39A12 protein, or a fragment, or derivative, or variant thereof, to a plurality of containers, and (ii) adding a detectable, preferably a fluorescently labelled compound or a plurality of detectable, preferably fluorescently labelled compounds to be screened for said binding to said plurality of containers, and (iii) incubating said SLC39A12 protein, or said fragment, or derivative, or variant thereof, and said detectable, preferably fluorescently labelled compound or detectable, preferably fluorescently labelled compounds, and (iv) measuring the amounts of compound, preferably its fluorescence, associated with said SLC
  • SLC39A12 translation product or a fragment, or derivative, or variant thereof into artificial liposomes as described in the present invention.
  • Said assay methods may be useful for the identification of novel compounds as well as for evaluating compounds which have been improved or otherwise optimized in their ability to bind to SLC39A12 protein, or a fragment, or derivative, or variant thereof.
  • the present invention provides a method for producing a medicament comprising the steps of (i) identifying a compound as a binder to a gene product of the gene coding for SLC39A12 proteins by the aforementioned binding assays and (ii) admixing the compound with a pharmaceutical carrier.
  • said compound may also be identifiable by other types of screening assays.
  • the present invention provides for a medicament obtainable by any of the methods according to the herein claimed screening assays.
  • the instant invention provides for a medicament obtained by any of the methods according to the herein claimed screening assays.
  • protein molecules being translation products of the gene coding for SLC39A12 and the use of said protein molecules (preferably having SEQ ID NO: 1 , or SEQ ID NO: 2, or SEQ ID NO: 3), or fragments, or derivatives, or variants thereof, as diagnostic targets for detecting a neurodegenerative disease, in particular Alzheimer's disease.
  • the present invention further features protein molecules being translation products of the gene coding for SLC39A12 and the use of said protein molecules (preferably having SEQ ID NO: 1 , or SEQ ID NO: 2, or SEQ ID NO: 3), or fragments, or derivatives, or variants thereof, as screening targets for agents, modulators, antagonists, agonists, reagents or compounds preventing, or treating, or ameliorating a neurodegenerative disease, in particular Alzheimer's disease.
  • the present invention features antibodies which are specifically immunoreactive with an immunogen, wherein said immunogen is a translation product of the SLC39A12 gene coding for SLC39A12 proteins (preferably having SEQ ID NO: 1 , or SEQ ID NO: 2, or SEQ ID NO: 3), or fragments, or derivatives, or variants thereof.
  • the immunogen may comprise immunogenic or antigenic epitopes or portions of a translation product of said gene, wherein said immunogenic or antigenic portion of a translation product is a polypeptide, and wherein said polypeptide elicits an antibody response in an animal, and wherein said polypeptide is immunospecifically bound by said antibody.
  • antibody as employed in the present invention, encompasses all forms of antibodies known in the art, such as polyclonal, monoclonal, chimeric, recombinatorial, anti-idiotypic, humanized, or single chain antibodies, as well as fragments thereof (see Dubel and Breitling, Recombinant Antibodies, Wiley-Liss, New York, NY, 1999).
  • Antibodies of the present invention are useful, for instance, in a variety of diagnostic and therapeutic methods, based on state-in-the-art techniques (see Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999 and Edwards R., Immunodiagnostics: A Practical Approach, Oxford University Press, Oxford, England, 1999) such as enzyme- immunoassays (e.g. enzyme-linked immunosorbent assay, ELISA), radioimmunoassays, chemoluminescence-immunoassays, Western-blot, immunoprecipitation and antibody microarrays. These methods involve the detection of translation products of the SLC39A12 gene, or fragments, or derivatives, or variants thereof.
  • enzyme- immunoassays e.g. enzyme-linked immunosorbent assay, ELISA
  • radioimmunoassays e.g. enzyme-linked immunosorbent assay, ELISA
  • said antibodies can be used for detecting the pathological state of a cell in a sample obtained from a subject, comprising immunocytochemical staining of said cell with said antibody, wherein an altered degree of staining, or an altered staining pattern in said cell compared to a cell representing a known health status indicates a pathological state of said cell.
  • the pathological state relates to a neurodegenerative disease, in particular to AD.
  • Immunocytochemical staining of a cell can be carried out by a number of different experimental methods well known in the art.
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 1 shows the identification of differences in the levels of SLC39A12 gene derived mRNA in human brain tissue samples from individuals corresponding to different Braak stages as measured and compared by GeneChip analyses. It indicates that the levels of the respective mRNA species correlate quantitatively with AD progression and thus are indicative for AD as measured by the neuropathological staging of brain tissue samples according to Braak and Braak (Braak staging).
  • cRNA probes of frontal cortex as well as of inferior temporal cortex each of 5 different donors with Braak stage 0 (C011 , C012, C026, C027, and C032), 7 different donors with Braak stage 1 (C014, C028, C029, C030, C036, C038, and C039), 5 different donors with Braak stage 2 (C008, C031 , C033, C034, and DE03), 4 different donors with Braak stage 3 (C025, DE07, DE11 , and C057), and 4 different donors with Braak stage 4 (P012, P046, P047, and P068) have been applied to an analysis of an Affymetrix Human Genome U133 Plus 2.0 Array respectively. Differences reflecting an up-regulation of the SLC39A12 gene progressively with Braak stages predominantly in inferior temporal tissue are shown.
  • Figure 3 shows the analysis of absolute levels of SLC39A12 gene derived mRNA in human brain tissue samples from individuals corresponding to different Braak stages indicative for AD as measured by quantitative RT-PCR and using statistical method of the median at 98%-confidence level (Sachs L (1988) Stat Vietnamese Methoden: Lor und Ausêt. Heidelberg New York, p. 60).
  • the data were calculated by defining control groups including subjects with Braak stages 0 to 2, which are compared with the data calculated for the defined groups with advanced AD pathology including Braak stages 3 to 4.
  • a substantial difference reflecting an up-regulation is shown in frontal as well as in inferior temporal cortices corroborating results from the GeneChip analysis.
  • Figure 4A discloses SEQ ID NO: 1 , the amino acid sequence of the human SLC39A12 protein (splice variant 1 , sv1 ) (UniProt primary accession number Q504Y0).
  • This SLC39A12 protein comprises 691 amino acids.
  • Figure 4C discloses SEQ ID NO: 3, the amino acid sequence of the human SLC39A12 protein (splice variant 3, sv3) (UniProt primary accession number Q5VWV8).
  • This SLC39A12 protein comprises 654 amino acids.
  • Figure 6C depicts SEQ ID NO: 9, the coding sequence (cds) of the human SLC39A12 sv3, comprising 1965 nucleotides, harbouring nucleotides 221 to 2185 Of SEQ ID NO. 6.
  • Figure 8 schematically charts the alignment of the SLC39A12 cDNA sequence SEQ ID NO: 4, the SLC39A12 coding sequence SEQ ID NO: 7 and both primer sequences used for SLC39A12 transcription level profiling (SEQ ID NO: 10, SEQ ID NO: 11 ). Sequence positions are indicated on the right side.
  • HKQ1 detects a double band migrating between approximately 70 to 90 kDa (arrow), probably reflecting the presence of different isoforms (sv1 , sv2, sv3) and/or different states of post-translational modifications of the SLC39A12 full-length protein.
  • This signal is weakly present in human brain extract (lane 1 ), whereas it is not detected in the negative control cell extract (lane 3). It is completely abolished by preincubation with the specific peptide.
  • the anti-myc antibody detects the SLC39A12 full-length protein as a double band migrating at -70-90 kDa (lane 8).
  • Figure 10 shows an immunohistochemical analysis of the production and localization of SLC39A12 protein in human brain tissue.
  • SLC39A12 protein is specifically immunodetected in the nuclei and cytoplasm of neurons and glia, as well as in the neuropil.
  • Affinity- purified polyclonal rabbit anti-SLC39A12 antiserum HKQ1 was used for indirect immunofluorescence staining. Depicted are immunofluorescence micrographs of acetone-fixed cryostat sections from a fresh-frozen post-mortem human temporal forebrain specimen.
  • Genotypes used were (1 ) w1118; (2) w; gmr-GAL4, UAS-TauP310L/+; (3) w; +/UAS-SLC39A12#4; gmr-GAL4, UAS-TauP310L/+; (4) w; +/UAS-SLC39A12#30; gmr-GAL4, UAS-TauP310L/+; (5) w; gmr-GAL4, UAS- TauP310L/UAS-SLC39A12#47.
  • Figure 13 shows a comparison of SLC39A12 mRNA level expressed by three independent SLC39A12 transgenic fly lines under the control of gmr-GAL4 by RT-PCR using SLC39A12 specific primers.
  • Genotypes used were (1 ) w; gmr-GAL4, UAS-TauP310L/+; (2) w; +/UAS- SLC39A12#4; gmr-GAL4, UAS-TauP310L/+; (3) w; +/UAS-SLC39A12#30; gmr- GAL4, UAS-TauP310L/+; (4) w; gmr-GAL4, UAS-TauP310L/UAS-SLC39A12#47.
  • Genotypes used were: (1 ) w; gmr-GAL.4, UAS-TauP310L/+; (2) w; +/UAS-SLC39A12#4; gmr-GAL.4, UAS-TauP310L/+; (3) w; +/UAS-SLC39A12#30; gmr-GAL.4, UAS-TauP310L/+; (4) w; gmr-GAL.4, UAS-TauP310L/UAS- SLC39A12#47.
  • FIG. 15 shows Western blot analysis of SLC39A12-expression in H4- neuroglioma cells stably co-expressing the Swedish Mutant APP.
  • SLC39A12 was myc-tagged at the C-terminus and introduced into tissue culture cells. Expression of SLC39A12 is driven by the CMV-promoter. Cells were harvested, lysed and subjected to Western Blot analysis using an antibody directed against the myc-epitope at a 1 :3000 dilution. In lane B bands running at approx. 85 kDa becomes visible. In the control cell line expressing an unrelated protein no corresponding band running at the same molecular weight is visible (lane A)
  • EXAMPLE 1 Identification and verification of Alzheimer's disease related differentially expressed genes in human brain tissue samples.
  • GeneChip microarray (Affymetrix) analyses were performed with a diversity of cRNA probes derived from human brain tissue specimens from clinically and neuropathologically well characterized individuals. This technique is widely used to generate expression profiles of multiple genes and to compare populations of mRNA present in different tissue samples.
  • mRNA populations present in selected post-mortem brain tissue specimens frontal and inferior temporal cortex
  • Tissue samples were derived from individuals that could be grouped into different Braak stages reflecting the full range between healthy control individuals (Braak 0) and individuals that suffered from AD signs and symptoms (Braak 4). Verification of the differential expression of individual genes was performed applying real-time quantitative PCR using gene-specific oligonucleotides. Furthermore specific differences between healthy and disease stages were analysed at the protein level using gene product specific antibodies for immunohistochemical analyses. The methods were designed to specifically detect differences of expression levels at early Braak stages, which is indicative for pathological events occurring early in the course of the disease. Thus, said genes identified to be differential are effectively implicated in the pathogenesis of AD.

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Abstract

L'invention traite d'un dérèglement du gène SLC39A12 et de ses produits protéiques chez des patients atteints par la maladie d'Alzheimer. A partir de cette découverte, l'invention décrit des méthodes de diagnostic et de pronostic de la maladie d'Alzheimer chez un individu et des méthodes permettant de déterminer si un individu présente un risque accru de développer la maladie d'Alzheimer. L'invention décrit également des méthodes thérapeutiques et prophylactiques de traitement et de prévention de la maladie d'Alzheimer et des troubles neurodégénératifs associés aux gène SLC39A12 et à ses produits géniques correspondants. L'invention concerne également des procédés de criblage destinés à moduler les agents des maladies neurodégénératives.
PCT/EP2006/062835 2005-06-01 2006-06-01 Proteines slc39a12 en tant que cibles diagnostiques et therapeutiques pour les maladies neurodegeneratives Ceased WO2006128902A1 (fr)

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EP2660600A4 (fr) * 2010-12-28 2015-05-06 Sumitomo Dainippon Pharma Co Ltd Médicament de diagnostic et procédé de diagnostic pour la maladie d'alzheimer
WO2017017446A1 (fr) * 2015-07-28 2017-02-02 Imperial Innovations Ltd Agents thérapeutiques

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EP2660600A4 (fr) * 2010-12-28 2015-05-06 Sumitomo Dainippon Pharma Co Ltd Médicament de diagnostic et procédé de diagnostic pour la maladie d'alzheimer
US10393757B2 (en) 2010-12-28 2019-08-27 Dainippon Sumitomo Pharma Co., Ltd. Diagnostic drug and diagnostic method for Alzheimer's disease
CN102174099A (zh) * 2011-01-28 2011-09-07 河北农业大学 一种阿尔茨海默症的靶位蛋白质及其编码基因和应用
WO2017017446A1 (fr) * 2015-07-28 2017-02-02 Imperial Innovations Ltd Agents thérapeutiques
US11248230B2 (en) 2015-07-28 2022-02-15 Ip2Ipo Innovations Limited Therapeutic agents

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